Method of enhancing efficacy of blood transfusions

ABSTRACT

A method of improving the efficacy of a blood transfusion into a subject is provided comprising administering a composition comprising an EAF PEGylated-blood protein into the subject, prior to, during, or subsequent to the blood transfusion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/794,978, filed Mar. 12, 2013, which claims the benefit of U.S.Provisional Patent App. No. 61/613,105, filed Mar. 20, 2012, thecontents of each of which are herein incorporated by reference in theirentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersR24-HL 064395 and R01-HL 062354 awarded by U.S. Public Health Service(Bioengineering Research Partnership) and W81XH1120012 awarded by the USArmy Medical Research and Material Command. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to bynumber in parentheses. Full citations for these references may be foundat the end of the specification. The disclosures of these publicationsand of all books, patents and patent application publications citedherein are hereby incorporated by reference in their entirety into thesubject application to more fully describe the art to which the subjectinvention pertains.

Treatment of blood losses usually proceeds in a sequence where theinitial reduction of blood volume is corrected using plasma expanders.Blood itself is used when the blood loss continues and extends beyondthe so called “transfusion trigger”. Outcomes in this process aresignificantly determined by the restoration of normal microvascularcirculation—characterized by the extent to which functional capillarydensity (FCD) returns to normal (1). FCD is determined as the number ofcapillaries per unit area of tissue observed with passage of red bloodcells (RBCs).

Normal FCD results from the adequate transmission of blood pressure tothe periphery and the absence of capillary obstructions due to capillarycollapse and abnormal blood cells. Moderate levels of colloidal plasmaexpansion and hemodilution up to about 50% exchange have no effects onFCD. However if bleeding follows hemodilution the conditions of theorganism are significantly affected by the extent of hemodilution andvolume loss, a situation described by Van der Linden and Vincent as“tolerance to hemorrhage following hemodilution,” although the effect ofdifferent types of hemodiluents have not been investigated (2-4)(5).

Crystalloid and colloidal-based plasma expanders are used in the initialphase of blood volume restoration. New hypotheses are emerging on therelative efficacy of colloid based plasma expanders with significantlydifferent biophysical properties, particularly regarding their viscosityand colloidal osmotic pressure (COP) (2-4). Clinically, human serumalbumin (HSA) is also used for plasma expansion in patients (15) buthydroxyethyl starch (HES) is currently the most common clinically usedcolloid (5-7). Polyethylene glycol (PEG) conjugated human serum albumin(PEG-Alb) (8) has yielded improved microvascular outcomes inexperimental resuscitation scenarios (9-14).

This invention provides a method of improving the efficacy of bloodtransfusions and of improving FCD following transfusions.

SUMMARY OF THE INVENTION

A method is provided of improving the efficacy of a blood transfusioninto a subject comprising administering a composition comprising aPEGylated-blood protein into the subject, wherein the PEGylated-bloodprotein is administered to the subject prior to, during, or subsequentto the blood transfusion into the subject.

Also provided is a PEGylated-blood protein for improving the efficacy ofa blood transfusion into a subject.

A method for treating a sickle cell disease in a subject who hasreceived, is receiving or will receive blood transfusion to treat thesickle cell disease comprising administering to the subject acomposition comprising PEGylated-blood protein or a compositioncomprising PEGylated-blood protein antioxidant conjugate, wherein thePEGylated-blood protein or PEGylated-blood protein antioxidant conjugateis administered to the subject prior to, during, or subsequent to theblood transfusion into the subject.

A method for reducing one or more lesions (or for reducing the extent ofdegradation or of oxygen-carrying capacity) resulting from, orassociated with, storage of a red blood cell-containing composition,blood, or a blood derivative intended for subsequent transfusion,comprising admixing the red blood cell-containing composition, blood, ora blood derivative with an amount of EAF PEGylated-blood protein with orwithout an antioxidant conjugated thereto and/or of a reactive oxygenspecies nanomaterial in an amount effective to reduce one or morelesions (or the extent of degradation or of oxygen-carrying capacity)resulting from, or associated with, storage.

Also provided is a composition comprising (i) red blood cells, (ii)blood, or (iii) a blood derivative intended for subsequent transfusion,admixed with an amount of EAF PEGylated-blood protein with or without anantioxidant conjugated thereto and/or of a reactive oxygen speciesnanomaterial, as described herein, effective to reduce lesions (or forreducing the extent of degradation or of oxygen-carrying capacity)otherwise resulting from storage thereof.

Further aspects of the invention are apparent from the followingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The experimental protocol. Time course of the acute isovolemicexchange transfusion/hemodilution with 4% PEG-Albumin, HES, or plasma,the two-step hemorrhage procedure, and resuscitation with either freshautologous or stored whole blood.

FIG. 2A-2C: Fresh autologous blood versus stored blood. Analysis withinthe same treatment group 4% PEG-Albumin (A), HES (B), or fresh plasma(C): P<0.05 relative to basal level (*), Hemodilution (†), 15% H (‡).Analysis between treatments at the same time point: P<0.05 (§). 4%PEG-Albumin (PEG-Alb); Fresh autologous blood (FB); Stored blood (SB).

FIG. 3: Fresh autologous/stored blood: 4% Peg-Albumin vs. HES vs.plasma. Analysis between treatments at the same time point of animalsresuscitated with fresh autologous (A) or stored (B) blood: P<0.05 (§).4% Peg-Albumin (Peg-Alb); Fresh autologous blood (FB); Stored blood(SB).

FIG. 4: Schematic representation of oligomerization of albumin bycombining two different extension arm chemistry, one introducing thiolat the distal end of the extension arm (butirimidyl moiety, aconservative mode) and the other introducing maleimide moiety at thedistal of the extension arm (caproylated protein, a non-conservativemode).

FIG. 5: Thiolation (extension arm chemistry)-mediated oligomerization ofalbumin using PEG bis-maleimide and limiting the intermolecularcrosslink between a pair of albumin molecules to one.

FIG. 6: Influence of EAF Bis maleimide PEG based oligomerization of EAFPEG albumin on the shear thinning effect.

FIG. 7: Shear thinning effect of EAF PEG Hb.

DETAILED DESCRIPTION OF THE INVENTION

A method is provided of improving the efficacy of a blood transfusioninto a subject comprising administering a composition comprising aPEGylated-blood protein into the subject, wherein the PEGylated-bloodprotein is administered to the subject prior to, during, or subsequentto the blood transfusion into the subject.

A method for treating a sickle cell disease in a subject who hasreceived, is receiving or will receive blood transfusion to treat thesickle cell disease comprising administering to the subject acomposition comprising PEGylated-blood protein or a compositioncomprising PEGylated-blood protein antioxidant conjugate, wherein thePEGylated-blood protein or PEGylated-blood protein antioxidant conjugateis administered to the subject prior to, during, or subsequent to theblood transfusion into the subject.

In a preferred embodiment, the composition is administered prior to thetransfusion, but at less than 30 mins, less than 1 hour, less than 2hours, less than 5 hours, less than 12 hours, or less than 24 hoursbefore the blood transfusion into the subject. In an embodiment, thecomposition is administered during the transfusion. In an embodiment,the composition is administered subsequent to the transfusion, but atless than 15 mins., less than 30 mins., less than 1 hour, less than 2hours, less than 5 hours, less than 12 hours, or less than 24 hourssubsequent to the blood transfusion into the subject.

In an embodiment, the PEGylated-blood protein is an extension arm (in aconservative or a non-conservative mode) facilitated (EAF)PEGylated-blood protein. In an embodiment, the composition comprises 4%hexaPEGylated-albumin. In an embodiment, the PEG of the compositioncomprising the PEGylated-blood protein is 5,000 mw PEG. In anembodiment, the PEG is selected from PEG-3000, PEG-5000, PEG-7500, PEG10,000, PEG-15,000, PEG-20,000, PEG-30,000, and/or PEG-40,000.

In an embodiment, the blood transfusion comprises blood or a bloodcomponent, and the blood or the blood component has been obtained from ablood donor more than two weeks prior to the transfusion. In anembodiment, the blood transfusion comprises blood or a blood component,and the blood or the blood component has been obtained from a blooddonor more than one month prior to the transfusion.

In an embodiment, the EAF PEGylated-blood protein is oligomerized EAFPEGylated-blood protein. In an embodiment, the oligomerized EAFPEGylated-blood protein is a linear polymer. In an embodiment, theoligomerized EAF PEGylated-blood protein is a globular polymer.

In an embodiment, the blood protein is a hemoglobin. In an embodiment,the blood protein is an albumin. In an embodiment, the blood protein ishexaPEGylated.

In an embodiment, the blood protein (with or without EAF PEGylation) iscovalently bonded to an antioxidant (an “EAF-PEGylated blood proteinantioxidant conjugate” or a “blood protein antioxidant conjugate”,respectively). In an embodiment, the blood protein is EAFpolynitroxylated.

In an embodiment, the methods further comprise administering to thesubject a reactive oxygen species nanomaterial as described.

In an embodiment, the methods further comprise super-perfusing the EAFPEGylated-blood protein, EAF PEGylated-blood protein antioxidantconjugate and/or reactive oxygen species nanomaterial.

In an embodiment, the subject (i) has suffered a hemorrhage; (ii) isundergoing surgery; (iii) has undergone surgery within the previous 30days; (iv) is suffering from an effect of a hemorrhagic shock; (v) haslost more than 15% of his or her blood volume within the last 48 hours;or (vi) has a sickle cell disease.

In an embodiment, the subject is in a hemodiluted state prior to theblood transfusion. In an embodiment, the composition administeredcomprises 1-10% EAF PEGylated-blood protein. Without being bound bytheory, in an embodiment the administered compound improves functionalcapillary density.

In preferred embodiments of the methods described herein, thePEGylated-blood protein, the PEGylated-blood protein antioxidantconjugate, or compositions comprising such are administered into thebloodstream of the subject. In an embodiment, such can be achievedintravenously or intra-arterially. In an embodiment, such can also beachieved by administering the PEGylated-blood protein, thePEGylated-blood protein antioxidant conjugate, or compositionscomprising such, as a component of the blood transfusion administered tothe subject.

Also provided is a PEGylated-blood protein for improving the efficacy ofa blood transfusion into a subject. In an embodiment, thePEGylated-blood protein is formulated to be administered to the subjectprior to, during, or subsequent to the blood transfusion into thesubject. In an embodiment, the PEGylated-blood protein is an extensionarm facilitated (EAF) PEGylated-blood protein. In an embodiment, thePEGylated-blood protein or (EAF) PEGylated-blood protein has anantioxidant conjugated thereto.

As used herein, a “blood transfusion” is the administration of aquantity of whole blood, or of one or more blood products directly orindirectly into the bloodstream a subject. In non-limiting examples,blood products include one or more of red blood cells, white bloodcells, plasma, clotting factors, and platelets. In an embodiment, thetransfusion comprises an autologous transfusion (i.e. the transfusionblood or blood product has previously been obtained from the samesubject). In a preferred embodiment, the transfusion comprises anallogeneic transfusion (i.e. the transfusion blood or blood product hasbeen obtained from a different subject of the same species). In anembodiment, the transfusion is performed after the “transfusion trigger”has been reached in the recipient subject. Blood transfusion inefficacycan lead to various complications due in part to the need to repeattransfusions, and inefficacy can be especially serious for critical-carepatients requiring rapid restoration of oxygen delivery. Insufficientefficacy can result from different causes, including blood product unitsdamaged by storage lesion—a set of biochemical and biomechanical changeswhich occur during storage. With red blood cells, this can decreaseviability and ability for tissue oxygenation. In an embodiment, thesubject receiving, the subject who will receive or the subject who hasreceived the blood transfusion, has not received a plasma expander. Inan embodiment, the subject receiving, the subject who will receive orthe subject who has received the blood transfusion, has received aplasma expander.

The “transfusion trigger” is the art-recognized critical point in thetreatment subject's status at which a therapy provider, usually aphysician, decides to transfuse a patient in order to achieve adequatetissue oxygenation.

Table 1 shows defined transfusion triggers (from the AmericanAssociation of Critical Care Nurses):

Source: RBC Infusion Platelet Infusion FFP Infusion Cyro InfusionAmerican Society Rarely for Rarely for Microvascular Consider for ofAnesthesiologists Hgb >10 g/dL PLT >100,000 bleeding present fibrinogenlevels <80 Guidelines for Usually for Usually for and PT or PTT is mg/dLto 100 mg/dL Blood Component Hgb <6 g/dL PLT <50,000 1.5 times normal orwhen levels can Therapy Decision based For PLT between In the absence ofnot be rapidly on risk for 50,000 and lab results: After obtainedcomplications 100,000 decision transfusion of 1 related to based ontotal blood volume inadequate assessment of risk Condemns use foroxygenation volume replacement Coffland & Shelton Symptoms, not PLT<50,000 Condemns use for Minimum Hgb and Hct, volume therapeutic shoulddictate replacement fibrinogen 50-100 transfusion mg/dL Symptomaticanemia Crosson — PLT <100,000 Only if PT and Fibrinogen <150 PTT >1.5times mg/dL normal After 10 u of RBCs Dennis (1992) — Condemns After 10u of RBCs — prophylactic use Bleeding times usu abnormal after 5 u RBCs;little value in determination PLT <100,000 Faringer et al HCT <30%Penetrating Only monitor PT Fibrinogen <100 (1993) trauma with low ForPT >1.5 times mg/dL PLT: delayed until normal microvascular bleeding isidentified Blunt trauma with low PLT: replace promptly Hurley Medical —Oozing and Initial: 2 u FFP — Center PLT <50,000 after 10 u RBCsFollowed by: 1 u FFP after each additional 5 u RBC Consider coagulationSpence Hgb alone should — — — not dictate transfusion Must understandphysiologic anemia Hgb = hemoglobin; Hct = hematocrit; PLT = platelets;PT = prothrombin time; PTT = partial thromboplastin time; FFP = fresh,frozen plasma; RBCs = red blood cells; Cryo = cryoprecipitate.

As used herein the “functional capillary density” (FCD) is the number ofcapillaries per unit volume of tissue permitting flow of RBCs.

As used herein a “blood protein” is a protein usually found in the bloodof the relevant (mammalian) subject, such as hemoglobin or albumin or,if specified, a derivative thereof, such as a hemoglobin derivativeshowing an improved or decreased different affinity for oxygen relativeto native hemoglobin. In an embodiment, the blood protein is ahemoglobin or an albumin, and includes isolated, purified or recombinantforms of each thereof. In an embodiment, the blood protein is a humanhemoglobin. In an embodiment, the blood protein is a human albumin.

In embodiments, the PEG may be PEG-3000 through PEG-40,000. In anembodiment, the PEG is selected from PEG-3000, PEG-5000, PEG-7500, PEG10,000, PEG-15,000, PEG-20,000, PEG-30,000, and/or PEG-40,000, whereinthe number refers to the average molecular weight of the PEG. In anembodiment, the PEG geometry may be linear, branched, star, or comb.

In an embodiment, the blood protein is PEGylated with 1 to 100 PEGmolecules, more preferably 2 to 24 PEG molecules, each attached via aflexible chemical linker (an extension arm). In a preferred embodiment,the blood protein is hexaPEGylated, with each of the PEG molecules beingattached via a flexible chemical linker (an extension arm).

PEGylated blood proteins, including albumins and hemoglobins, andmethods of synthesis thereof, are also described in U.S. Pat. No.8,071,546 and in U.S. Patent Application Publication No. US 2009-0298746A1 and in PCT International Publication No. WO 2011/106086, the contentsof each of which are hereby incorporated by reference.

As used herein, an “extension arm facilitated” (EAF) PEGylated proteinshall mean a protein having at least one PEG molecule attached theretovia a flexible chemical linker (an extension arm). In an embodimentwhere a plurality of PEG molecules are each attached via flexiblechemical linkers, the extension arm facilitated PEGylated protein iscapable of a lower packing density of the attached PEG chains in thePEG-shell than the packing density in the protein to which the PEGchains are linked. In an embodiment, the extension arm (i.e. theflexible small molecular weight aliphatic linker in between the proteinand the PEG) is ˜1 nm in length.

In an embodiment, a EAF PEGylated protein of the invention may be one ofthe following:

where n is an integer from 1 through 100 and X is a blood protein, suchas a hemoglobin or an albumin, and wherein m is the number of ethyleneglycol monomers. PEGs of from 1,000 to 40,000 daltons are preferred. Them value can be chosen to provide PEGs of from 1,000 to 40,000 daltons.

In an embodiment, the EAF PEGylated blood protein can further comprisean antioxidant. In an embodiment, the antioxidant is a thiol, thioether,glutathione, curcumin, N-acetyl methionine, or SOD mimetic (e.g., tempol(1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine) or a proxyl orpolysulfide). In an embodiment, the EAF PEGylated blood protein canfurther comprise one or more of the following molecules:

wherein X is the blood protein, (or its EAF PEGylated form, or theirpolymeric forms, e.g. nanomaterials), and wherein n is an integer from 1through 100, and where the antioxidant molecule is any of the speciesreferred to hereinabove in the tempol shown in this structure or inplace of the tempol shown.

Also provided is a method for reducing one or more lesions (or forreducing the extent of degradation or of oxygen-carrying capacity)resulting from, or associated with, storage of a red bloodcell-containing composition, blood, or a blood derivative intended forsubsequent transfusion, comprising admixing the red bloodcell-containing composition, blood, or a blood derivative with an amountof EAF PEGylated-blood protein antioxidant conjugate and/or of areactive oxygen species nanomaterial in an amount effective to reduceone or more lesions (or the extent of degradation or of oxygen-carryingcapacity) resulting from, or associated with, storage. In an embodiment,the stored red blood cell-containing composition, blood, or bloodderivative is intended for subsequent transfusion to a subject. In apreferred embodiment, the subject is a human. In non-limiting examples,the red blood cell-containing composition, blood, or blood derivative isstored for one week, two weeks, three weeks, or four weeks. Alsoprovided is a composition comprising (i) red blood cells, (ii) blood, or(iii) a blood derivative intended for subsequent transfusion, admixedwith an amount of EAF PEGylated-blood protein antioxidant conjugateand/or of a reactive oxygen species nanomaterial, as described herein,effective to reduce lesions (or for reducing the extent of degradationor of oxygen-carrying capacity) otherwise resulting from storagethereof.

Also provided is a kit comprising an amount of EAF PEGylated-bloodprotein antioxidant conjugate and/or of a reactive oxygen speciesnanomaterial, as described herein, effective to reduce lesions (or forreducing the extent of degradation or of oxygen-carrying capacity)otherwise resulting from storage of (i) red blood cells, (ii) blood, or(iii) a blood derivative intended for subsequent transfusion, andinstructions for use to reduce one or more of such lesions.

In an embodiment, the EAF PEGylated-blood protein antioxidant conjugateis EAF P5K6 Albumin Tempol 12 or (EAF P5K6 Albumin Tempol 12)_(n)wherein n is a positive integer from 1 to 40. In an embodiment, the (EAFP5K6 Albumin Tempol 12)_(n) is used and wherein n is a positive integerfrom 4 to 40.

Also provided is a PEGylated-blood protein oligomer having the formula(EAF P5K6 blood protein)_(n), where n is a positive integer of from 1 to100. In an embodiment, the PEGylated-blood protein oligomer issynthesized according to the method set forth in FIG. 5. In anembodiment of the PEGylated-blood protein oligomer, n is a positiveinteger of from 4 to 40.

Also provided is a PEGylated-blood protein oligomer comprising a centralblood protein with 1 to 4 further blood protein molecules attachedthereto, each through an EAF linker. In an embodiment, thePEGylated-blood protein oligomer is synthesized according to the methodset forth in FIG. 4.

In an embodiment of the PEGylated-blood protein oligomers, eachoccurrence of the blood protein is an albumin or is a hemoglobin. In anembodiment the PEGylated-blood protein oligomer comprises both albuminand hemoglobin. In an embodiment the PEGylated-blood protein oligomerfurther comprises one or more PEGylated-blood protein antioxidantconjugate(s) or PEGylated-blood protein SOD mimetic conjugate(s). In anembodiment, the blood protein of the PEGylated-blood protein antioxidantconjugate, is albumin.

Also provided is a PEGylated-blood protein for improving the efficacy ofa blood transfusion into a subject. In an embodiment, thePEGylated-blood protein is formulated to be administered to the subjectprior to, during, or subsequent to the blood transfusion into thesubject. In an embodiment, the PEGylated-blood protein is an extensionarm facilitated (EAF) PEGylated-blood protein. In an embodiment, thePEGylated-blood protein is further conjugated to an antioxidant. In anembodiment, each occurrence of the blood protein is an albumin or ahemoglobin.

The subject of the methods herein can be a mammal. In differentembodiments, the mammal is a mouse, a rat, a cat, a dog, a horse, asheep, a cow, a steer, a bull, livestock, a primate, or a monkey. Themammal is preferably a human.

Where a numerical range is provided herein, it is understood that allnumerical subsets of that range, and all the individual integerscontained therein, are provided as part of the invention. Thus, a bloodprotein which comprises from 1 to 100 PEG molecules includes the subsetof PEGylated blood proteins which comprise 1 to 50 PEG molecules, thesubset of PEGylated blood proteins which comprise 10 to 75 PEGmolecules, and so forth, as well as a PEGylated blood protein whichcomprises 6 PEG molecules, a PEGylated blood protein which comprises 7PEG molecules, a PEGylated blood protein which comprises 8 PEGmolecules, up to and including a PEGylated blood protein which comprises100 PEG molecules.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Example 1

The effects of PEG-Alb as an adjunct or aid to blood transfusion itselfare not known. Herein, hamster plasma was used to compare PEG-albuminwith albumin because plasma is approximately 70% albumin. Hamster plasmais a convenient source of hamster albumin, avoiding potentialimmunological effects of colloids from different species. In thiscontext, PEGylation is proposed to render the albumin proteinimmune-invisible since water immobilized near the protein by PEGpresumably isolates active elements from immune receptors (16).Additionally, the effects of the condition of the blood used in thetransfusion process were investigated, since blood storage is known toadversely affect microvascular perfusion. Whether the outcome ofresuscitation with a blood transfusion in a hemodiluted subject isdependent in any way on the colloid used prior to blood transfusion wasexplored, as was whether the nature of the type of colloid can overcomesome of the shortcomings resulting from longer blood storage times.

The hamster chamber window model was used to compare the microvasculareffects of hemodilution with fresh plasma, HES, or a 4% PEG-Alb followedby hemorrhagic shock treated by restoration of oxygen carrying capacitywith fresh blood or 2-week-stored blood.

Example 2

Preparation of PEGylated blood protein, their oligomeric forms(nanomaterials) and their antioxidants: The EAF PEGylation of the bloodproteins and their oligomeric forms is carried out as discussedpreviously (16, 19). EAF polynitroxylation of PEGylated blood proteinsis also carried out just as EAF PEGylation except that maleimido phenylurethane of PEG is replaced by tempol, and minim fast flow filtrationwas used to take out the excess reagents, 2-IT and maleimido Tempol.

Oligomerization of Albumin and EAF-PEGylation of the Oligomeric albumin:Oligomerization of the albumin is another desirable approach for themodulation of the solution properties, particularly viscosity ofPEG-albumin. Studies by Marcos Intaglietta and Amy Tsai using alginatehave exposed the correlation between the viscosity and plasma expansion,in particular vasodilation. Nonetheless, the PEG-albumin, with asignificantly lower viscosity than alginate with the combination of goodCOP, makes it a better plasma expander than alginate. Besides dextran 70that is isoviscous EAF P5K6 Albumin is not only vasodilatory, it alsoinduced endothelial NO production just as dextran 500. However, it isnot known how EAF P5K6 Albumin (and EAF P5K6 Hb) induces endothelial NOproduction, like dextran 500 does, even though the its viscosity nearlythree times lower.

Generating the oligomeric forms of albumin, and EAF PEGylation,generates, for example [(EAF P5K6) Albumin]n, where n refers to numberof the monomeric units in the polymer. The increased viscosity isexpected to even further increase endothelial NO production. For furtheroligomers of albumin, two types of novel approaches for oligomerizationbased on the extension arm chemistry developed earlier, which chemistryis considered as a click chemistry using thiol maleimide reaction as thebasic reaction. In a first approach two different extension arms arecombined on two different albumin molecules, in one molecule of albuminextension arms have thiolated proteins, and in a second moleculeextension arms have maleimide at the distal end. The thiolated albuminis generated by reaction the protein with 2-IT as described previously.This step of the reaction is controlled to generate Alb with one or twoor three or four thiol groups on the albumin molecule. These molecularspecies with different levels thiols on their surface are reacted withan albumin derivative generated by reacting with one equivalent ofε-maleimido caproyl sulfo succinimide ester (sulfo EMCS). Albumin withsingle copy of maleimide caproyl moiety conjugated (ε-maleimido on the nchemistry (conservative and nonconservative heterobifunctional reagentsto introduce intermolecular crosslinks). This is schematically presentedin FIG. 4. Using iminothiolane, albumin is generated with one, two,three and four extrinsic thiols per albumin monomer. These are trappedas mixed disulfide with thiopyridyl. Since thiopyridyl groups areattached to the extrinsic thiols, the albumin molecules with differentnumber of thiol groups will have different charges as the thiopyridylmixed disulfides. Thus, these can be purified by conventional ionexchange chromatography.

Another class of monofunctional maleimide derivative of albumin isgenerated by reacting albumin with ECMS (see FIG. 4) or sulfo EMCS. Freethiols of the purified thiolated albumins are generated by releasing themixed disulfide using TCEP (or glutathione), and mixed withmaleimide-carrying albumin in amounts slightly more than one equivalent(per thiol) of the respective thiolated albumins. TCEP does not reactwith maleimide. The number of maleimide-carrying albumin that can beconjugated to the thiolated albumin is dictated by the number of thiolsin the thiolated albumin. To quench the olgimerization reaction, lowmolecular weight thiol compounds are added to the reaction mixture totrap the excess maleimide-carrying albumin. Size exclusionchromatography will be used, if needed, to purify the oligomers ofdifined molecular size (i.e. defined number of molecules). It isexpected that a pentamer can be generated by this approach. To generateoligomers with higher numbers of albumin units, a second cycle ofthiolation and reaction with maleimide functionalized albumin isperformed. The albumin derivative so generated has one central albuminmolecule and sequentially placed layers of outer albumin. Accordingly,when it is EAF PEGylated, almost all the PEG chains conjugated theretoare surface-decorating the outer most layer of albumin molecules. Theseoligomers can be described as globular oligomers of EAF PEG Albumin.

In a second strategy, thiolated albumins are oligomerized usingbifunctional PEG based maleimides. The principle is essentially the sameas discussed above and is schematically presented below (FIG. 5). Sincethe design strategy introduces one crosslink between a pair of albuminmolecules the resultant oligomer is an elongated molecule. Such arereferred to as ellipsoidal oligomers of albumin. On EAF PEGylation, eachmolecule is PEGylated as there will be no steric hindrance to the accessof the PEG reagent, a macromolecular reagent.

The oligomers generated by the first approach are expected to be morerigid (compact) than the oligomers generated by the second approach. Dueto the flexibility of the molecule, the viscosity is expected to behigher with the later, similarly the pseudoplasticity (shear thinningeffect). Very pure reagents with 4, 8, and 12 oxyethylene units areavailable commercially. Purified oligomers of albumin will be subjectedto EAF-PEGylation to optimize the molecular, biophysical and chemicalproperties just as we have planned with albumin as discussed earlier.

Covalent attachment of Antioxidants (Nitroxides or thiols or methionine)to albumin and/or to PEG-albumin: For covalent attachment of nitroxidesto albumin the extension arm chemistry can be used. Both conservativeand non-conservative extension arm chemistry protocols are selected asdesired. Conservative extension arm chemistry is preferred when largernumbers of antioxidants are to be attached to albumin. Albumin or PEGalbumin is thiolated using either iminothiolane or succinimidyl estersof thiol containing aliphatic acid (commercially available). Nitroxides,TEMPOL and PROXYL are non-limiting examples of two antioxidants that canbe used for generating this class of compounds. Albumin or PEG albuminis thiolated and then reacted with appropriate maleimide oriodoacetamide derivatives of the desired antioxidants. The approach isto optimize the conjugation chemistry so that the adducts generatedexhibit maximum level of enzyme mimetic activity as well as longerhalf-life. The earlier approach has resulted in extensive electrostaticmodification of the molecular surface of albumin, and this might bedeemed to result in a low half-life for this product.

The enzyme mimetic activity of antioxidants of TEMPOL class (sixmembered ring group) is nearly two orders of magnitude higher than thatof five membered series (PROXYL). Since nitroxides can recycle, thelevel of the antioxidant enzymem superoxide dismutase mimetic needed isexpected to be very limited. The level of these compounds conjugated toalbumin or PEG-albumin can be adjusted to minimize the toxicity fromthese antioxidants. Extension arm chemistry makes it easy to quantitateand control the level of enzyme mimetics covalently bound to albumin aswell as its PEGylated product.

In addition to the nitroxide class of albumin and PEG-albuminantioxidant adducts, another class of albumin antioxidant adducts can beproduced that carry extrinsic thiol functions. The extension armchemistry used to introduce thiols for PEGylation is the approach usedhere. Homocysteinylation of albumin can also be used, in view of itsreported high reductive potential. A third class of albumin antioxidantconjugates is albumin with multiple copies of methionine or N-acetylmethionine conjugated.

The extension arm chemistry developed introduces a thiosuccinimidomoiety into molecule for each extension arm engineered. This is athioether, and these are in principle catalase activity centers as thesedegrade hydrogen peroxide form reversible sulfoxides that are hydrolyzedor reduced by methionine sulfoxide reductase present in the tissues inthe in vivo situations. Additional polysulfides are conjugated using theEAF bis maleimide PEG approach to increase the catalase mimeticactivity, when needed, again using Extension Arm Chemistry. Theseclasses of molecules generated from Hb and albumin are thus oxygencarrying and non-oxygen carrying plasma expanders with reactive oxygenspecies scavenging activity. These properties coupled with thepseudoplasticity of these, make these an unique class of reactive oxygenspecies scavenging activity molecules exhibiting supra perfusionaryactivity.

Materials and Methods

Animal preparation: Studies were performed in golden Syrian malehamsters (Charles River Laboratories, Boston, Mass.), weight range of56-71 g. The Guide for the Care and Use of Laboratory Animals (USNational Research Council, 2010) was followed for animal handling.Experiments were approved by the University of California, San DiegoInstitutional Animal Care and Use Committee. The window chamber modelwas utilized for microvascular studies in unanesthetized hamsters.Chamber implantation and vascular catheterization surgeries wereperformed under general anesthesia, pentobarbital 50 mg/kg orketamine/xylazine cocktail 200/10 mg/kg i.p. injections, as previouslydescribed (17, 18). Following chamber implantation, animals recoveredfor a minimum two day period prior to catheterization. Animals wereanesthetized for carotid artery and jugular vein catheter implantation(polyethylene-50), following microscopic chamber assessment in order torule out edema, bleeding, or signs of infection Animals were enteredinto the study one to two days following catheterization surgery.

Systemic parameters: Mean arterial blood pressure (MAP) and heart rate(HR) were continuously monitored using a Biopac system (MP 150; BiopacSystems, Inc., Santa Barbara, Calif.) excluding blood sampling,hemodilution, and hemorrhage periods, where the arterial catheter was inuse. Systemic hematocrit (Hct) was measured from heparinizedmicrocapillary arterial blood collection, following centrifugation.Baseline systemic parameter requirements for inclusion of the animal inthe study were: MAP>80 mmHg, HR>320 beats/min, and systemic Hct>40%.

Functional Capillary Density (FCD): FCD was determined in 10 stepwisevertically successive microscopic fields using 20× magnification.Capillaries were considered functional in having at least one transitingRBC, during a 30 sec observation period. FCD (cm⁻¹) can be defined asthe total length of RBC-perfused capillaries divided by the surface areaof tissue in which they are observed (18). Initial tissue fields werechosen by a distinguishing anatomical feature to allow quick andaccurate recognition during repeated measurements.

Plasma expander fluids and experimental groups: Experiments were carriedout in 3 groups of hamster (n=12 each group) each group corresponding toa plasma expander used for hemodilution (PEG-Alb, HES or plasma). Eachplasma expander group was further divided into two groups (n=6 in eachsubdivided group) corresponding to the type of blood used inresuscitation following hemorrhage: one group received fresh autologousblood and the other group was treated with stored allogeneic wholeblood. Animals were randomized into both group divisions.

Albumin PEGylation has been previously described (13, 19). An extensionarm facilitated (EAF) PEGylated blood protein can be used. For example,an extension arm facilitated protocol can be used to PEGylate analbumin. In a non-limiting example, lyophilized preparations of albumin(Sigma Aldrich, St. Louis, Mo.) are subjected to cold (4° C.) EAFPEGylation for overnight at a protein concentration of 0.5 mM in thepresence of 5 mM 2-IT (for thiolation of the protein) using 10 mMmaleimidophenyl PEG 5 kDa (custom synthesized). Under these conditionson an average six to seven copies of PEG 5 kDa chains are conjugated tothe protein. The hexaPEGylated albumin thus generated can be purifiedthrough tangential flow filtration and concentrated to a 4 gm % solutionwith respect to albumin (2 gm % solution with respect to PEG; it is a 6gm % solution with respect to EAF PEG albumin calculated based on themolecular mass of EAF PEG albumin to be 95 to 100 kDa) and stored at−80° C.

The EAF PEG Albumin and EAF PEG Hb generated as described above are verydistinct as compared to the products referred to Maleimide PEG modifiedAlbumin (MPA) and Maleimide PEG modified Hb (MP) prepared by Sangart.MPA and MP4 are generated using: (a) Two-step version (protein isthiolated first and them mixed with maleimide as a second step of thereaction) of the EAF PEGylation platform and not the one-step version(thiolation and PEGylation carried out simultaneously) of EAF PEGylationdiscussed above; (b) Maleimido propyl PEG 5K and not Maleimido phenylurethane of PEG 5K discussed above; (c) Baxter Albumin (a material readyto be used for transfusion), a processed human derived albumin was usedfor MPA, the process used involved the heating of the human derivedalbumin at 60° C. for 10 hrs. EAF PEG Albumin discussed here isgenerated using high purity human serum albumin that has not beensubjected to the heating process; (d) unchromatographed human Hb(lysate) was used for the preparation of MP of Sangart, whereas Hb A₀purified from the lysate by Q-Sepharose chromatography is used for thepreparation of EAF PEG Hb discussed in the present study.

HES as used is a starch molecule of low molecular weight and with a lowdegree of molar substitution (Voluven; Fresenius-Kabi, Graz, Austria)(20).

Fresh plasma was obtained the same day of experiments from a donorhamster. Whole blood was collected in citrate and then centrifuged toobtain the plasma. The physical properties of the plasma expanders usedare given in Table 2.

TABLE 2 Properties of PEG-Albumin, HES, and plasma. PEG-Albumin HESPlasma Source Synthetic/Human Synthetic Hamster Concentration (%) 4 6 —Viscosity (cP) 2.2 2.1 1.2 Average molecular weight (kDa) 96 130 —Suspending fluid Phosphate buffer Saline — Degree of substitution — 0.40—

Collection and storage of whole blood and plasma: For fresh autologousblood collection, blood was withdrawn from the carotid artery catheterand was collected in 5 ml syringes containing citrate phosphate dextroseadenine-1 anticoagulant solution (Fenwal, Inc., Lake Zurich, Ill.,CPDA-1). The amount of CPDA-1 solution used was 0.14× total blood volume(BV) withdrawn. For stored blood, blood was withdrawn from the carotidartery catheter of a donor hamster and was collected into a 5 ml syringecontaining CPDA-1 (0.14× total BV=4.56 ml). The blood was thentransferred under sterile conditions to a sterile preservative freevacutainer tube and immediately placed in 4° C. storage for a 14-15 dayperiod.

Fresh plasma collection. Fresh plasma was obtained the same day as itwas used from a donor hamster and collected in citrate. Donor blood wascentrifuged, and plasma was separated from RBCs.

Experimental setup and acute isovolemic exchange transfusionhemorrhagic-shock and resuscitation protocol; The experimental procedurewas the same for all 6 animal groups. Unanesthetized animals were placedin a restraining tube, stabilized by securing the tube and the chamberto a plexiglass stage. The plexiglass stage holding the animal wasplaced on an intravital microscope (BX51WI; Olympus, New Hyde Park,N.Y.) equipped with a 20× objective (LUMPFL-WIR, numerical aperture 0.5;Olympus). Animals were given 15-30 min to adjust to the tube environmentbefore baseline measurements were taken (MAP, HR, FCD, and Hct).

Following baseline measurements, an acute anemic state was induced by a20% of BV (estimated as 7% of individual body weight) hemodilution,through an isovolemic exchange transfusion with either 4% PEG-Albumin,HES, or fresh plasma in citrate using a dual syringe pump (model 33syringe pump; Harvard Apparatus; Holliston, Mass.). The plasma expanderwas infused into the jugular vein catheter and blood was withdrawn fromthe carotid artery catheter at a rate of 100 μl/min. Animals were thengiven a 30 min stabilization period, during which FCD was measured.Systemic parameters were measured subsequent to the 30 min period, priorto the start of the two-step hemorrhage procedure. Following thestabilization period animals were subjected to a two-step hemorrhageprocedure. The volume of each hemorrhage step was a calculatedpercentage of the animal's BV. Step one and two were 40% and 15% of BV,respectively, each carried out in a 5 min period, with a 20 min waitingperiod given between the first and second step of hemorrhage.Resuscitation was initiated after 10 min with a 62.7% of BV bloodtransfusion using either fresh autologous or stored whole blood.Resuscitation volume (62.7%) included 55% (of BV) of hemorrhaged wholeblood, the balance being anticoagulant.

Fresh autologous blood was maintained at room temperature untilresuscitation. Stored blood was removed from storage and allowed to warmto room temperature 30 min prior to resuscitation. Results wereevaluated at 60 min after transfusion at which time the experiment wasterminated. FCD and systemic parameters were assessed as shown in theexperimental time table (FIG. 1).

Statistical analysis: Results are presented as mean±standard deviation,unless otherwise noted. Data are presented as absolute values and/or asvalues relative to baseline values. All measurements were compared withbaseline levels obtained prior to the start of experimental procedures.The same capillary fields were followed in order to enable directcomparisons to their baseline levels, allowing for more robuststatistics for small sample populations. For repeated measurements,within groups, time-related changes were assessed by ANOVA fornonparametric measurements (Kruskal-Wallis), and when appropriate posthoc analyses performed with the Dunn's multiple comparison test. Changesbetween group measurements were assessed using the Mann-Whitney test.All statistics were calculated with computer software (Prism 4.0,GraphPad, San Diego, Calif.). Changes were considered statisticallysignificant if p<0.05.

Results

Animals studied throughout the experimental procedures completed thehemodilution phase with a MAP change no greater than 10 mmHg, indicatinga successful exchange transfusion.

Hematocrit: Hct for all animal groups was decreased followinghemodilution as expected and then again following the two-stephemorrhage procedure, with significant decreases resulting in all groupscompared to both baseline and hemodilution values (Table 2). Hcts wererestored to post-hemodilution levels, not being significantly different,after the 60 min recovery period with no significant differences betweengroups resuscitated with fresh autologous blood. Animals resuscitatedusing stored blood increased Hct following resuscitation, returning topost-hemodilution values, however, remaining significantly decreasedcompared to baseline levels. Transfusion of stored blood caused asignificant increase in Hct 60 min post infusion for all plasma expanderpre-treatments (p<0.05, Table 3).

TABLE 3 Changes in hematocrit due to hemodilution, hemorrhage, andresuscitation. Plasma Resus- Time Point Measurement Ex- citation PostPost 15% 60 min Post pander Blood Hemodilution HemodilutionResuscitation PEG- Fresh 38.2 ± 5.7  22.9 ± 1.5* 33.8 ± 2.3* Albautologous (n = 6) Stored 36.7 ± 2.4* 25.0 ± 0.5*  38.2 ± 2.3*^(a) (n =6) HES Fresh 40.0 ± 2.9*  26.7 ± 1.4*^(a) 35.5 ± 2.4* autologous (n = 6)Stored 40.9 ± 1.5* 27.5 ± 1.5*  41.8 ± 1.6*^(b) (n = 6) Plasma Fresh37.8 ± 1.1* 25.5 ± 0.8* 34.8 ± 1.2* autologous (n = 6) Stored 38.5 ±1.0*  24.2 ± 2.2*^(c)  40.3 ± 4.6*^(cd) (n = 6) Baseline hematocrit (n =36): 48.0 ± 2.2%. Analysis within treatment groups: p < 0.05 *vs.baseline. Analysis between treatments at the same time point: p < 0.05^(a)vs. PEG-Alb Fresh blood; ^(b)vs. HES Fresh blood; ^(c)vs. HES Storedblood; ^(d)vs. Plasma Fresh blood. 4% Peg-Albumin (Peg-Alb);Hydroxyethyl starch (HES).

Mean arterial pressure. Hemodilution caused a non-significant reductionof MAP relative to baseline. MAP decreased significantly afterhemorrhage in all groups (p<0.05, Table 4). Additionally, MAPsignificantly decreased following hemorrhage relative to hemodilutionpressures (p<0.05), apart from animals in the HES/stored blood group.Baseline MAP levels were restored for all groups 60 min postresuscitation, the effect being greater for the group treated withstored blood (p<0.05, Table 4).

TABLE 4 Effect of hemodilution, hemorrhage and resuscitation on bloodpressure. Plasma Resus- Time Point Measurement Ex- citation Post Post15% 60 min Post pander Blood Hemodilution Hemodilution ResuscitationPEG- Fresh,  106.8 ± 11.2 40.9 ± 9.6*^(†) 94.4 ± 7.8  Alb autologous (n= 6) Stored 104.0 ± 8.7 37.7 ± 3.2*^(†)  116.1 ± 14.2^(‡a) (n = 6) HESFresh 108.5 ± 8.8  40.7 ± 14.7*^(†)  99.4 ± 11.3 autologous (n = 6)Stored 105.6 ± 3.5  40.6 ± 10.6* 111.1 ± 11.6^(‡) (n = 6) Plasma Fresh103.1 ± 5.7  32.6 ± 6.9*^(†a) 95.0 ± 7.1^(‡) autologous (n = 6) Stored108.1 ± 9.9 35.6 ± 7.8*^(†) 110.4 ± 21.6^(‡) (n = 6) Baseline MAP (n =36): 112.8 ± 9.6 mmHg. Analysis within treatment groups: p < 0.05 *vs.baseline; ^(†)vs. hemodilution; ^(‡)vs. 15% H. Analysis betweentreatments at the same time point: p < 0.05 ^(a)vs. PEG-Alb Fresh blood.4% Peg-Albumin (Peg-Alb). Hydroxyethyl starch (HES).

Heart rate: Hemodilution and hemorrhage decreased HR, which was returnedto near normal values by blood transfusion to an extent depending on theplasma expander used during hemodilution. The 4% PEG-Alb groups hadsignificantly diminished HRs due to hemorrhage compared to baseline(p<0.05, Table 5). The HES animal groups only showed a significant HRvariation in the fresh autologous blood resuscitated group. There wereno significant differences between the HRs of HES fresh autologous andstored blood resuscitated groups. Similarly, HRs of plasma hemodilutedanimals resuscitated with fresh autologous blood attained a significantdifference between the post recovery period and hemorrhage values, whileHRs of plasma animals resuscitated with stored blood were significantlydifferent following hemorrhage compared with baseline and hemodilution(p<0.05). Differences between plasma fresh autologous and stored bloodresuscitated animal group HRs were not significant (Table 5).

TABLE 5 Effect of hemodilution, hemorrhage and resuscitation on heartrate. Plasma Resus- Time Point Measurement Ex- citation Post Post 15% 60min Post pander Blood Hemodilution Hemorrhage Resuscitation PEG- Fresh483.2 ± 9.9  331.8 ± 54.5*^(†) 475.1 ± 12.3^(‡) Alb autologous (n = 6)Stored 475.7 ± 21.2 302.7 ± 30.6*^(†) 433.6 ± 65.2^(‡) (n = 6) HES Fresh485.1 ± 15.3 354.0 ± 39.7^(†)  487.0 ± 25.0^(‡) autologous (n = 6)Stored 469.5 ± 40.8 331.8 ± 48.9   457.2 ± 60.1  (n = 6) Plasma Fresh461.4 ± 31.8 329.0 ± 35.5    459.9 ± 13.9^(‡ab) autologous (n = 6)Stored 470.1 ± 23.8 311.4 ± 36.5*^(†) 457.6 ± 68.6  (n = 6) Baseline HR(n = 36): 464.8 ± 49.1 beats/min. Analysis within treatment groups: p <0.05 *vs. baseline; ^(†)vs. hemodilution; ^(‡)vs. 15% H. Analysisbetween treatments at the same time point: p < 0.05 ^(a)vs. PEG-AlbFresh blood; ^(b)vs. HES Fresh Blood. 4% Peg-Albumin (Peg-Alb).Hydroxyethyl starch (HES).

Blood gases: PO₂ levels for both fresh and stored blood resuscitation inall animal groups were significantly increased compared to baselinelevels, following hemorrhage. PO₂ levels remained elevated 60 min postresuscitation, compared to baseline. However, only the PEG-Albumin andHES animal group's PO₂ levels remained significantly increased. Theplasma animal groups returned to baseline levels (Table 6).

Base excess for all animal groups was significantly decreased comparedto baseline, following hemorrhage. Resuscitation with both fresh andstored blood effectively restored base excess values for all groups,with the exception of HES fresh blood, 60 min post resuscitation.

TABLE 6 Effect of hemorrhage and resuscitation on arterial PO₂ and baseexcess. Plasma Resuscitation Time Point Measurement Expander BloodParameter Hemodilution 15% H R60 PEG-Alb Fresh autologous PO₂ (mmHg)61.6 ± 5.8  108.8 ± 8.1*  75.8 ± 8.7*  (n = 6) Base Excess 5.3 ± 1.6−5.5 ± 3.6* 6.6 ± 1.2^(‡) Stored (n = 6) PO₂ (mmHg) 66.5 ± 6.5  113.5 ±9.3*   76.1 ± 10.9* Base Excess 4.2 ± 1.8 −4.4 ± 2.9* 5.2 ± 2.2^(‡) HESFresh autologous PO₂ (mmHg) 67.0 ± 5.9  132.0 ± 3.2*^(a ) 72.9 ± 8.0* (n = 6) Base Excess 4.2 ± 2.4 −12.4 ± 5.5*  4.6 ± 3.3  Stored (n = 6)PO₂ (mmHg) 64.9 ± 4.0  121.6 ± 7.2*   80.0 ± 15.6* Base Excess 5.3 ± 2.0−9.0 ± 3.7* 5.2 ± 2.1^(‡) Plasma Fresh autologous PO₂ (mmHg) 60.1 ± 13.4125.1 ± 4.9*  67.0 ± 10.6  (n = 6) Base Excess 6.5 ± 1.8 −10.8 ± 2.4* 5.9 ± 2.2^(‡) Stored (n = 6) PO₂ (mmHg) 64.1 ± 6.5  123.2 ± 13.9* 69.2 ±14.2  Base Excess 8.2 ± 1.3 −8.0 ± 3.8* 7.9 ± 1.4^(‡) Baseline (n = 36):PO₂ 56.5 ± 5.8 mmHg and Base Excess 5.8 ± 2.0. Analysis within treatmentgroups: p < 0.05 *vs. baseline; ^(‡)vs. 15% H. Analysis betweentreatments at the same time point: p < 0.05 ^(a)vs. PEG-Alb Fresh blood.Hemorrhage (H) and 60 min post-resuscitation (R60).

Functional Capillary Density: There was a consistent decrease in FCDfollowing hemodilution, hemorrhage, and 60 min post resuscitation (FIG.2). FCD did not change in the HES/fresh autologous blood group followinghemodilution. Hemorrhage significantly reduced FCD in all groups(p<0.05, FIG. 2) Animals pre-treated with HES and resuscitated withfresh autologous blood had significantly decreased FCD compared to thestored blood group (p<0.05, FIG. 2B). However, this difference was notmaintained through the 60 min recovery period. Only the HES pre-treatedgroup transfused with fresh autologous blood animals significantlyincreased FCD subsequent to hemorrhage (p<0.05, FIG. 2B).

Fresh autologous blood resuscitated animal groups, hemodiluted witheither 4% PEG-Alb or plasma, had significantly increased FCD bycomparison following resuscitation using stored blood (p<0.05, FIGS. 2 Aand C). However, only 4% PEG-Alb/fresh autologous blood resulted insignificantly increased FCD, 60 min post resuscitation, compared to posthemorrhage values (p<0.05, FIG. 2A).

FIG. 3 compares FCD for the plasma expander groups, divided according tothe final use of fresh autologous or stored blood resuscitation. Fourpercent PEG-Alb maintained FCD during hemorrhage and produced thehighest FCD 60 min after resuscitation (p<0.05, FIG. 3A).

A finding of this study is that the type of colloid used in the initialhemodilution of blood volume restitution affects the outcome FCD whenblood is transfused in correcting the continuation of hemorrhage. Theeffect is significant, following resuscitation with fresh blood, 60minutes after the blood transfusion, exhibiting a trend common for bothfresh and stored blood whereby the best result is obtained when 4%PEG-Alb is used initially and the worst result is for fresh plasma, withHES being intermediate. The trend is statistically significant for freshblood.

The same trend in the effect of the plasma expanders were observed forboth fresh and 2-week stored blood. However, fresh blood yielded aconsistently higher FCD upon transfusion by comparison to stored blood,an effect consistent with previous findings on the effects of storage onRBC properties (21). Results consistent with these were found in thestudy of Gonzalez et al. (22) who subjected rats to a 10% blood lossthat were subsequently treated with fresh blood and 1-week and 2-weeksold stored blood. In this study the principal effect is reported to besignificant 4 hr after transfusion, consisting in approximately an 8%decrease in FCD, with no difference between one and 2-week storage. Thisrather small effect is probably a consequence of the significantlysmaller transfusion performed in their study, 10% of blood volume vs.55% in this study.

Base excess was the same in all groups after blood transfusion. Howeverthe recovery of base excess, i.e., the change between hemorrhage (H %)and after transfusion (R60) was greater for the plasma and HEShemodilution. This in part reflects the lesser decrease of base excesswith PEG-Alb hemodilution due to the better FCD during hemorrhage (FIG.3).

The differences in systemic outcome between fresh and stored blood areevidenced by blood pressure being significantly elevated for the latter,by comparison with fresh blood. This is probably due to vasoconstrictioninduced by hemoglobin being liberated from RBCs during storage and fromhemolysis due to increased cell fragility of the cell in the storedblood, as evidenced by the presence of plasma hemoglobin in the range of0.1-1.7 mg/dl. Notably, differences in pre-treatment were not apparentfrom the measurement of MAP and HR.

The type of plasma expander used in the initial treatment of bloodlosses, simulated in this study by performing a 20% hemodilution, causedeffects on FCD not discernible by systemic observations (MAP or HR),which were practically the same for both types of blood transfusion asshown in Tables 3 and 4. However, there were significant difference inmicrovascular conditions between the best (4% PEG-Alb and fresh wholeblood) and worst (Fresh plasma and stored whole blood) scenarios. Thisinteraction between blood storage times and pre-existing bloodcomposition appear to be primarily related to how the initial plasmaexpansion affects microvascular function, prior to subsequent bloodlosses and transfusion, since the trend is already present during theshock period when the plasma expander is on board. In this context FCDis significantly higher during the shock period in animals previouslyhemodiluted with 4% PEG-Alb than when pre-treated with either HES orplasma.

Transfusion with fresh RBCs leads to a better outcome than using storedblood. One of the earliest investigation of this effect in conjunctionwith resuscitation in hemorrhagic shock was reported by Collins andStechenberg (23), who used a model of severely stressed rats. The modelconsisted in exchange transfusing 90% of rat's blood volume with eitherfresh blood or 14-20 day stored blood at different hematocrits (17, 25,and 34%), inducing an initial state of anemia, causing hemorrhage (˜50%of blood volume) and resuscitating with the same blood originallyexchanged. This study showed that a difference in survival between freshand stored blood was only evident for the anemic subjects and attributedthis difference to the reduction of oxygen supply to the tissue, fromthe reduction in Hct and the increased oxygen affinity due to thedepletion of 2, 3-DPG in the stored blood.

The present study and related results from microvascular studies suggestthat the results of the Collins and Stechenberg experiment and similarstudies are more specifically related to the effects on capillary flowper se rather than oxygen supply. Kerger et al. (24) showed thatextended untreated hemorrhage survival correlated with the maintenanceof a threshold FCD, independently of tissue oxygen, substantiatingprevious reports of the association between survival and improvedperfusion in skeletal muscle (25). Cabrales et al. (26) reported thatincreased oxygen affinity in anemia improves microvascular function andSakai et al. (27) showed that high oxygen affinity hemoglobin improvestissue oxygenation in anemia. Taken as a whole, these and other resultssuggest that the organism and tissue survive a considerable reduction ofHct provided that FCD is maintained. In this scenario, however, theintroduction of stored RBCs has the potential of inducing negativeeffects because of the reduction of FCD probably due to their reducedflexibility (28) causing capillary obstruction. The associatedhemolysis, liberating hemoglobin in plasma, may be an additional factorsince vasoconstriction can reduce capillary pressure (29).

The clinical significance of effects found in subcutaneous/skeletalmuscle tissue cannot be compared to that of internal tissues nor doesits microcirculation fully represent that of internal organs such as thekidney, heart, or, viscera. However, it was found that a correlationexisted between microvascular hemodynamics responses in the tissue ofthe window chamber and major organs (2). Thus the model is a compromisebetween observing microhemodynamic effects in an unexposed intacttissue, without anesthesia, and studying responses in the major organswhere microhemodynamic measurements cannot be made.

PEG-Alb was originally developed previously (30), but was notextensively investigated until recently. PEG-Alb is a comparativelylarge molecule that immobilizes a significant amount of water on itssurface. It has a relatively high COP (42 mmHg at 4% concentration),which limits its concentration in the circulation. As a consequence itspotential viscogenic effect is reduced; however, its physiologicaleffect remains similar to that of a high viscosity plasma expander,which significantly improves microvascular function in extreme anemicconditions. Four percent PEG-Alb is not particularly viscous, a propertythat is significantly lowered as the effective viscosity in plasma isdecreased by dilution. The beneficial effects of 4% PEG-Alb appear to bedue to a combination of factors, including hemodilution which reducedoverall blood viscosity and increased blood flow, a modest increase inplasma viscosity, and increased nitric oxide (NO) bioavailability (31)by mechanotransduction in the endothelium.

This study focuses on events occurring at the initial stages of shockresuscitation. In previous studies it was shown that microvascular andtissue dysfunction during shock treatment with HES occurs 15 minutesafter the initiation of treatment (10). In the present study it is shownthat this dysfunction is only partially corrected by the restoration ofoxygen carrying capacity because the critical restoration of functionalcapillary density is limited and dependent on blood storage. It shouldbe noted that shock treatment is associated with a secondary decrease inmicrovascular flow that occurs within the first 20 to 40 min of the postresuscitation period, reported to occur in the microcirculation of theskeletal muscle, the ileum and the kidney progresses (32, 33),therefore, the observation point at 1 hour after initiation of treatmentshould include the effects of secondary decompensation.

In conclusion, the use of stored blood consistently yields decreasedmicrovascular function by comparison with fresh blood, an effectreflected by many studies comparing the effects of blood storage (34).Outcome in terms of microvascular function when a blood transfusion ismade following initial blood volume restoration with plasma expanders isshown to depend on the type of plasma expander, results being optimalfor 4% PEG-Alb, and progressively worse for HES and homologous plasma.Thus, the microvascular outcome following a blood transfusion isdependent on how the organism and the microcirculation react to theplasma expander used prior to a blood transfusion. In addition, themicrocirculation responds differentially depending on the extent ofblood storage. As a corollary it appears possible to diminish themicrovascular effects due to blood storage by improving pre-transfusionmicrovascular function.

Example 3

In further experiments, extreme hemodilution to a hematocrit (Hct) of11% with polyethylene glycol conjugated albumin (PEG-Alb) was comparedto the use of Dextran 6% 70 kDa and 6% Dextran 500 kDa in a 3 isovolemicsteps protocol. Effects were analyzed by measuring the rheologicalproperties of the plasma expander blood mixture; a model analysis of thedistribution of wall shear stress in the microvessels, measurement ofincreased NO bioavailability above baseline levels in the vessel wall(measured using microelectrodes) and measurement of cardiac output.Plasma expansion with PEG-Alb caused a state of supra-perfusion withcardiac output 40% above baseline and significantly increased NO vesselwall bioavailability and lowered peripheral vascular resistance. Thiscondition arises because the mixture of blood and PEG-Alb is shearthinning, which as shown by a mathematical modeling of the distributionof shear stress becomes maximal at the vessel wall (wall shearstress—“WSS”) and minimal in the blood vessel core. An analysis wasperformed using the Quemada model for describing the rheology of bloodand quantifiying viscosity of blood as a function of Hct and shear rate,accounting for the presence of a cell sparse plasma layer near thevessel walls with blood plasma modeled as a Newtonian fluid. The neteffect of the hemodilution using the comparatively low viscosity shearthinning PEG-Alb plasma expansion was a reduction of overall bloodviscosity, an increased WSS and therefore endothelial NO production.These changes act synergistically, significantly increasing cardiacoutput and perfusion due to lowered overall peripheral vascularresistance.

Results for perivascular NO measurements: Six animals were entered intothis study for the measurement of NO, and all animals tolerated thehemodilution protocol without visible signs of discomfort. Two animalswere used as controls to insure that the system calibration and animalpreparation was the same as previous control measurements (36). PEG-Albperivascular NO levels have not been previously published. Physiologicalconditions and the rheological properties of blood after a level 3exchange are presented in Table 7. For comparison, data on low viscosityhemodilution using Dextran 70, and high viscosity hemodilution usingDextran 500 from the previous study by Tsai et al. (35) is alsoincluded. The principal finding from these measurements is that theincreased perfusion found in conditions of extreme hemodilution usingPEG-Alb is associated with increased arteriolar and venular perivascularNO. These NO concentrations were significantly greater than in thecontrol and when extreme hemodilution was performed Dextran 70. The NOconcentrations were the same as those found using Dextran 500.

TABLE 7 Hemodilution with low viscosity Dextran 70, high viscosityDextran 500 and PEG-Alb. Control Dextran 70 Dextran 500 PEG-Alb Hct, %49.3 ± 1.8 11.1 ± 0.9 11.0 ± 0.6 11.2 ± 0.7 MAP, mmHg 103 ± 6  64 ± 8 87± 6 79 ± 7 Heart rate, bpm 414 ± 35 418 ± 41 453 ± 38 460 ± 31 Cardiacoutput, 17.8 ± 1.6 14.2 ± 1.9 19.6 ± 3.5 24.2 ± 1.6 ml/min Base-excess,mmol/l  3.2 ± 1.5 −4.6 ± 2.6  0.8 ± 1.6  1.3 ± 1.4 Plasma viscosity, cP 1.2 ± 0.1  1.4 ± 0.2  2.2 ± 0.2  1.3 ± 0.1 (#) Blood viscosity, cP  4.1± 0.4  2.1 ± 0.2  2.8 ± 0.2  1.8 ± 0.1 (#) Peripheral 5.8 4.5 4.4 3.3resistance, (§) (#) Viscosity measured in Brookfield cone and plateviscometer at 200 sec⁻¹; (§) Ratio of mean arterial blood pressure (MAP)and cardiac output.

Molecular and solution properties of (EAF-P5K6)_(n): Without being boundby theory, it is proposed that the unique effects of EAF P5K6 Albumin interms of increasing efficacy of blood transfusion is presumably eitherrelated to the supra-perfusion achieved by the presence of this materialin the plasma or the ability of this molecule to induce endothelial NOproduction that increase the bioavailability of NO in the plasma. Theaspects of supraperfusion and/or bioavailability of NO are linked to theviscosity and the shear thinning behavior of PEG Albumin, and this couldbe enhanced by the oligomerization of EAF-P5K6-albumin. One suchmaterial has been generated, and the molecular solution properties ofthis material is presented in Table 8.

TABLE 8 Molecular solution of properties of Oligomerized EAF PEG AlbuminMolecular Molecular PEG Shell PEG5K PEG Shell Radius Volume Volume chaindensity COP Viscosity (nm) (nm3) (nm3) Number1 (dal/nm3) (mmHg) (cP) HSA4.0 268 — — — 13.2 1.1 PEGylated HSA 2 8.1 2226 1958 9.0 23.0 53.2 2.76PEGylated HSA3 7.7 1912 1644 ~6 18.2 28.3 1.83 Oligo-HSA  14.4 4 12511 —0 — — — PEGylated 23   50979 38468 7.6   4.9 4 28.5 3.7 Oligo-HSA1Determined by NMR analysis. 2. Prepared at the reaction condition HSA +2-IT + MalPEG5K(0.5:10:10 mM) reacted in PBS at 4 C. for 4 hrs.3Prepared at the reaction condition HSA + 2-IT + MalPEG5K(0.5:5:5 mM)reacted in PBS at 4 C. for 6 hrs. 4. Assumed the average molecularnumber in each oligomer is about 5.

The oligomerization of the EAF PEG Albumin into the ellipsoidal form hasincreased the molecular dimensions to 25 nm as compared to about 7 nmfor EAF PEG Albumin with six copies of PEG 5K. The viscosity of EAF PEGAlbumin as expected, and also resulted in the reduction of the colloidaloncotic pressure of the material as a result of the decrease in thenumber of particles in solution for given weight of PEG albumin; thecolloidal oncotic pressure is a colligative property of the solution.This is also good for the application of material in the presentapplications because the auto-transfusion of the water from the tissuesto the vascular space will be reduced.

The oligomerization process has been optimized for producing EAF P5K6Alb oligomers with molecular radius ranging from 25 nm to 100 nm, andthese should now be addressed as nanoparticles rather than molecules.

Pseudoplasticity [Shear thinning effect of (EAF P5K6 Alb)_(n)]: Theshear thinning effect of a 4 gm % solution of [EAF P5K6 Alb]n has beencompared with that of EAF-P5K6-Alb and uncross-linked Hb in FIG. 6.Except for an increase in the viscosity of EAF PEG albumin onoligomerization, the general pattern of the shear thinning effectremains essentially unchanged.

The shear thinning effect of SP-P5K6-Hb: The shear thinning effect ofEAF HexaPEGylated Hb has also been established and presented in FIG. 7.The shear thinning effect of EAF PEG Hb appears to be more significantthan seen with EAF hexaPEGylated Albumin. The molecular dimensions ofthe protein core and PEG shell in EAF P5K6 Hb and EAF P5K6 Albumin arevery distinct, and this appears to be responsible for this difference.

Influence of Polynitroxylation of EAF P5K6 Hb on the shear thinningeffect: The polynitroxylation of EAF hexaPEGylated Hb has also beenelucidated. The polynitroxylation has been carried out using EAchemistry and the maleimido Tempol. Engineering six copies of Tempol togenerate EAF P5K6 Hb-Tempol 12 has no influence on the shear thinningeffect of EAF P5K6 Hb. Thus EAF PEG Albumin antioxidants and EAF PEG Hbantioxidants can be used to increase the efficacy of blood transfusionwith an engineered antioxidant activity.

Comparison of the in vitro antioxidant Activity of EAF PEG AlbuminTempol 6 and EAF PEG Albumin Tempol 12: The antioxidant activity of theantioxidant activity of Tempol conjugated to albumin and Hb are comparedin Table 9. The results show that conjugation of tempol to HSA by EAchemistry increases the catalytic activity of Tempol. However in thecase of EAF PEG Hb, the influence of EA chemistry on the catalyticactivity of tempol conjugated is much smaller, but the intrinsicactivity of tempol molecule on Hb is considerably higher than that onAlbumin. We consider that this may be the intrinsic peroxidase activityassociated with heme centers.

TABLE 9 The O2— induced reduction rate of Hb-Tempol and HSA-Tempolnitroxides. Tempo removing (rate/min/per rate (NT/min) mole Tempol) EAFP5K6-Hb-EAF-T6 −0.278 −0.046 EAF P5K6-Hb-T1 −0.036 −0.036 HSA-Tempo12−0.041 −0.003 HSA-EAF-Tempol 12 0.096 0.008

EA Chemistry-bis maleimiide PEG based conjugation of polysulfide to EAFPEG Alb Tempol Adducts to enhance the Efficacy of these as ReactiveOxygen Species Scavenging particles (molecules) exhibiting supraperfusionary activity: The antioxidant activity of the Tempol on EAF PEGAlbumin is considerably lower than that on the EAF PEG Hb. Thisapparently reflects some kind of synergy between the conjugated tempoland the heme center of EAF PEG Hb. In an attempt to increase theantioxidant activity of Tempol conjugated on EAF PEG Alb, new catalasemimetic activity will be engineered by conjugation of block polymers ofpolypropylene sulfide. EA chemistry will be adopted for this. Thepolypropylene sulfide will be monofunctionally functionalized asmaleimide by reacting with bis maleimide PEG (short chains 3 to 4 unitsof propylene oxide). The bloc polymers of propylene sulfide will bebetter than the thioether in the NEM modified thiols groups of extensionarms that has been discussed. By modulating the size of the oligomer(EAF-P5K6-Albumin)n, before subjecting it to thiolation mediatedpolynitroxylation, and conjugation with functionalized poly propylenesulfide. The previously designed macromolecular antioxidants along withthe new novel macromolecular antioxidants designed here, will serve asReactive Oxygen Scavenging nano-materials (ROS nanoparticles) with supraperfusion and will be excellent materials for antioxidant therapies andto reduce the storage lesions in RBC and to increase the efficacy oftransfusion when the stored blood is used for transfusion.

Multimodal Therapy for Sickle Cell Disease (SCD): The studies haveidentified EAF P5K6 Albumin and EAF P5K6 Albumin Tempol 12 as themolecules (or other derivatives of this class) as antioxidanttherapeutic agents for sickle cell disease. Transfusion is an acceptedtherapeutic approach for the treatment of the SCD. Consistent with theresults presented here, the combining the two approaches for anefficient therapy for SCD is desirable.

DISCUSSION

One significance of these findings is that they show why PEG-Albhemodilution produces a state of supra-perfusion. This occurs becauseblood is diluted, lowering blood viscosity in high shear rate zones ofthe circulation, like the heart and major vessels; while apparentviscosity and WSS increase in the microcirculation promoting theproduction of NO by the endothelium and vasodilatation. An extreme caseof this effect is the conversion of the fluid in the blood vessel coreinto a solid piston (maximum viscosity at zero shear rate), with a thinperipheral lubricating layer between the piston and cylinder. Thiscombination of effects, which should also be operational in the heartmuscle, allows the heart to maintain blood pressure and increase cardiacoutput, leading to the highly beneficial effect found in using PEG-Albplasma expansion.

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1. A method of improving the efficacy of a blood transfusion into asubject comprising administering a composition comprising aPEGylated-blood protein into the subject, wherein the PEGylated-bloodprotein is administered to the subject prior to, during, or subsequentto the blood transfusion into the subject.
 2. A method for treating asickle cell disease in a subject who has received, is receiving or willreceive blood transfusion to treat the sickle cell disease comprisingadministering to the subject a composition comprising PEGylated-bloodprotein or a composition comprising PEGylated-blood protein antioxidantconjugate, wherein the PEGylated-blood protein or PEGylated-bloodprotein antioxidant conjugate is administered to the subject prior to,during, or subsequent to the blood transfusion into the subject.
 3. Themethod of claim 1, wherein the PEGylated-blood protein is an extensionarm facilitated (EAF) PEGylated-blood protein.
 4. The method of claim 2,wherein the composition comprises 4% hexaPEGylated-albumin.
 5. Themethod of claim 2, wherein the blood transfusion comprises blood or ablood component, and the blood or the blood component has been obtainedfrom a blood donor more than two weeks prior to the transfusion.
 6. Themethod of claim 5, wherein the blood transfusion comprises blood or ablood component, and the blood or the blood component has been obtainedfrom a blood donor more than one month prior to the transfusion.
 7. Themethod of claim 3, wherein the EAF PEGylated-blood protein isoligomerized EAF PEGylated-blood protein.
 8. The method of claim 7,wherein the oligomerized EAF PEGylated-blood protein is a linearpolymer.
 9. The method of claim 7, wherein the oligomerized EAFPEGylated-blood protein is a globular polymer.
 10. The method of claim2, wherein the blood protein is a hemoglobin.
 11. The method of claim 2,wherein the blood protein is an albumin. 12-16. (canceled)
 17. Themethod of claim 2, wherein the subject (i) has suffered a hemorrhage;(ii) is undergoing surgery; (iii) has undergone surgery within theprevious 30 days; (iv) is suffering from an effect of a hemorrhagicshock; (v) has lost more than 15% of his or her blood volume within thelast 48 hours; or (vi) has a sickle cell disease.
 18. The method ofclaim 1, wherein the subject is in a hemodiluted state prior to theblood transfusion.
 19. The method of claim 2, wherein the compositioncomprising the EAF PEGylated-blood protein is administered to thesubject prior to the blood transfusion.
 20. The method of claim 2,wherein the composition administered comprises 1-10% EAF PEGylated-bloodprotein.
 21. The method of claim 2, wherein the PEG of the compositioncomprising the PEGylated-blood protein is 5,000 mw PEG.
 22. A method forreducing one or more lesions resulting from, or associated with, storageof a red blood cell-containing composition, blood, or a blood derivativeintended for subsequent transfusion, comprising admixing the red bloodcell-containing composition, blood, or a blood derivative with an amountof EAF PEGylated-blood protein with or without an antioxidant conjugatedthereto and/or of a reactive oxygen species nanomaterial in an amounteffective to reduce one or more lesions resulting from, or associatedwith, storage.
 23. The method of claim 22, wherein the EAFPEGylated-blood protein antioxidant conjugate is EAF P5K6 Albumin Tempol12 or (EAF P5K6 Albumin Tempol 12)_(n) wherein n is a positive integerfrom 1 to
 40. 24. The method of claim 23, wherein the (EAF P5K6 AlbuminTempol 12)_(n) is used and wherein n is a positive integer from 4 to 40.25-37. (canceled)